Abstract Circulating small regulatory RNAs (sRNAs) are short non-coding RNAs (typically ~19-25nt in size). They mediate a broad spectrum of biological processes through regulation of gene expression. Our experimental evidence indicates that serum levels of miRNAs (one form of sRNA) change considerably, the vast majority increasing with age. The ability of circulating sRNAs to travel among tissues enables them to transmit signals and regulate a broad spectrum of biological functions. sRNAs exist in a variety of RNase-insensitive ribonucleoprotein or lipid complexes, or are encapsulated inside different types of extracellular vesicles. Consequently, in contrast to messenger RNA, sRNAs are protected from extracellular RNases and are measurable and stable in samples stored for decades. Despite numerous recent developments, we are far from understanding the role of sRNAs in aging. An understanding of their role in aging mammals, and in humans in particular, is still very limited due to the increased complexity and longer life-spans of mammals compared with invertebrates. This project leverages existing human sample resources from three completed NIH-funded studies (EPESE, STRRIDE and CALERIE), to discover and validate longevity-associated miRNAs in humans. Our preliminary analysis of 175 circulating microRNA--in the NIA-funded Duke Established Populations for Epidemiologic Studies of the Elderly (Duke EPESE) community-based cohort of elders--identified 32 differentially expressed circulating miRNAs (p<0.05) associated with longevity; in all cases, their concentrations at baseline were higher in long-term survivors (>10 years) compared with age, sex and race matched but short-term survivors (<2 years); a subset of these miRNAs predicted longevity independent of age, gender, race and functional status. The Duke EPESE cohort was aged 71 and older at the time of blood sampling and now has 25 years of longitudinal mortality data (through 2016) with which to address key questions about sRNAs and longevity in humans. sRNA discoveries in Duke EPESE will be validated in plasma and muscle samples from completed human clinical trials of relevance to longevity that investigated the health-promoting effects of exercise (STRRIDE cohort) and caloric restriction (CALERIE cohort). A human three-dimensional muscle tissue organ system will be used to understand their mechanisms of action (with and without simulated exercise and caloric restriction), by testing sRNA mimics and inhibitors. Our preliminary analyses of 7 of our top longevity-related miRNA in this model system demonstrated production and secretion of all of them by muscle and statistically significantly increased secretion of two of them with simulated muscle exercise. Together our approach will permit us to determine if sRNAs associated with longevity are favorably modulated in tissue and blood in humans by exercise and/or caloric restriction, and if they appear to mediate any of the observed health benefits of these interventions. The totality of the data (generated in vivo and in vitro), will be systematically examined to identify pathways of sRNA action in humans and profiles of sRNA that could serve as biomarkers to predict longevity status.